Hydrophobic ceragenin compounds and devices incorporating same

ABSTRACT

A hydrophobic cationic steroidal anti-microbial (ceragenin) compound forms an amphiphilic compound having a hydrophobic sterol face and a hydrophilic cationic face. Medical devices can be made incorporating the ceragenin compound.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/602,499, filed Jan. 22, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/554,957, filed Jul. 20, 2012, which claims thebenefit of U.S. Prov. App. No. 61/572,714 filed Jul. 20, 2011, and61/642,431, filed May 3, 2012, the disclosures of which are incorporatedherein in their entirety.

BACKGROUND OF THE INVENTION

1. The Field of the Invention

The present invention relates to hydrophobic ceragenin compounds anddevices incorporating the hydrophobic ceragenin compounds. The ceragenincompounds have hydrophobic substituents that give the compounds arelatively high C Log P value that allow the compounds to benon-covalently bonded to polymeric materials.

2. The Relevant Technology

Ceragenin compounds, also referred to herein as cationic steroidal antimicrobial compounds (CSA), are synthetically produced small moleculechemical compounds that include a sterol backbone having various chargedgroups (e.g., amine and cationic substituents) attached to the backbone.The backbone can be used to orient the amine or guanidine groups on oneface, or plane, of the sterol backbone. For example, a scheme showing acompound having primary amino groups on one face, or plane, of abackbone is shown below in Scheme I:

Ceragenins are cationic and amphiphilic, based upon the functionalgroups attached to the backbone. They are facially amphiphilic with ahydrophobic face and a polycationic face. Without wishing to be bound toany particular theory, the anti-microbial ceragenin compounds describedherein act as anti-microbial agents (e.g., anti-bacterials,anti-fungals, and anti-virals). It is believed, for example, that theanti-microbial ceragenin compounds described herein act asanti-bacterials by binding to the outer cellular membrane of bacteriaand other microbes and inserting into the cell membrane forming a porethat allows the leakage of ions that are critical to the microbe'ssurvival and leading to the death of the affected microbe. In addition,the anti-microbial ceragenin compound described herein may also act tosensitize bacteria to other antibiotics. For example, at concentrationsof the anti-microbial ceragenin compounds below the correspondingminimum bacteriostatic concentration, the ceragenins cause bacteria tobecome more a susceptible to other antibiotics by increasing thepermeability of the outer membrane of the bacteria.

The charged groups are responsible for disrupting the bacterial cellularmembrane, and without the charged groups, the ceragenin compound cannotdisrupt the membrane to cause cell death or sensitization.

BRIEF SUMMARY

The present invention relates to ceragenin compounds that are relativelyhydrophobic despite having a hydrophilic cationic face. The highhydrophobicity has been found to have a surprising and unexpectedability to bond with polymers and then selectively release from thepolymeric materials to kill microbes.

In one embodiment, hydrophobic ceragenin compounds disclosed herein have(i) a sterol structure comprising four fused carbon rings; (ii) at leastone cationic substituent attached to each of at least three of the fourfused carbon rings so as to form an amphiphilic compound having ahydrophobic sterol face and a hydrophilic cationic face; (iii) at leastone hydrophobic substituent attached to at least one of the fused carbonrings; and (iv) wherein the CSA compound has a C Log P value of at least6.0.

The C Log P value is achieved by selecting a proper hydrophobicsubstituent(s) in combination with proper cationic substituents. Thecationic substituents and hydrophobic substituent(s) are selected togive a C Log P value of 6.0 or greater, 6.25 or greater, 6.5 or greater,7.5 or greater, or even 10 or greater. In order to achieve the desired CLog P value, greater hydrophobicity in the hydrophobic substituent isneeded when cationic substituents with less hydrophobicity are used.

The high C Log P value allows the compounds to be non-covalently bondedto polymers that have hydrophobic moieties. For example, the hydrophobiccompounds described herein can be non-covalently bonded to a hydrogelmaterials. The hydrophobic bonding allows for ceragenin compounds toassociate with the polymer while having minimal impact on the ability tokill microbes.

Surprisingly and unexpectedly, it has been found that by non-covalentlybonding the ceragenin to a polymeric material usinghydrophobic/hydrophilic interactions, the hydrophobic ceragenin compoundcan selectively release from the polymer in the presence of microbes,thereby having a killing affect at lower concentration than one wouldpredict and over an extended period of time. This is in contrast tostudies done with covalently bonded ceragenins where immobilizationimpeded kill rates beyond the initial exposure. The ability of thehydrophobic ceragenin compounds to selectively release from a polymer tokill microbes is highly desirable and a surprising and unexpectedresult.

In addition, it has been found that the ceragenins as used in thepresent invention surprisingly kill harmful microbes preferentially overnormal flora, which means that the ceragenins can be used at lowerconcentrations compared to other antimicrobials while achieving the sameor better effectiveness. This feature avoids many of the deleteriouseffects of prior art antimicrobials, many of which tend to kill the“good microbes.”

The hydrophobic ceragenin compounds can be incorporated into or formedinto medical devices such as medical devices to be implanted into ahuman or other animal. For example, the hydrogels can be coated on amedical device or incorporated into a polymeric product such as anophthalmic product. The medical devices incorporating the hydrophobiccompounds can controllably release ceragenin compound in a concentrationsufficient to meet regulatory requirements for maximum bacterial loadsover weeks or even months.

These and other features of the present invention will become more fullyapparent from the following description and appended claims, or may belearned by the practice of the invention as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by reference to specific embodiments thereof which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only illustrated embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1A illustrates example ceragenin compounds with a C Log P valueless than 6.5;

FIG. 1B illustrates example ceragenin compounds with a C Log P valuegreater than 6.5;

FIG. 2 is a schematic representation of a substrate with a polymericcoating;

FIG. 3 is a graph showing elution of a ceragenin from a hydrogel inphosphate buffered saline;

FIG. 4 is a graph showing elution of a ceragenin from a hydrogelfollowing autoclaving;

FIG. 5 is a graph showing elution of a ceragenin from a hydrogel inphosphate buffered saline and tryptic soy broth;

FIG. 6 is a graph showing elution of a ceragenin from a hydrogel inbuffer and 10⁶ CFU of S. aureus; and

FIG. 7 is a graph showing elution of a ceragenin from a hydrogel inbuffer and 10⁶ CFU of P. aeruginosa.

DETAILED DESCRIPTION I. Hydrophobic Ceragenins

In one embodiment, hydrophobic ceragenin compounds disclosed herein have(i) a sterol structure comprising four fused carbon rings; (ii) at leastone cationic substituent attached to each of at least three of the fourfused carbon rings so as to form an amphiphilic compound having ahydrophobic sterol face and a hydrophilic cationic face; (iii) at leastone hydrophobic substituent attached to at least one of the fused carbonrings; and (iv) wherein the CSA compound has a C Log P value of at least6.0, at least 6.25, at least 6.5, at least 7.5, or at least 10.

The C Log P value is achieved by selecting a proper hydrophobicsubstituent(s) in combination with proper cationic substituents. Thecationic substituents and hydrophobic substituent(s) are selected togive a C Log P value of 6.0 or greater, 6.25 or greater, 6.5 or greater,7.5 or greater, or even 10 or greater.

The ceragenin compound may have a structure as shown in Formula I:

Where:

rings A, B, C, and D form a fused ring system and at least one of the Rgroups on 2 or 3 of the 4 four fused rings has a cationic substituent.The other R groups on FIG. I can have a variety of differentfunctionalities, thus providing the ceragenin compound with the desiredhydrophobic properties.

In a preferred embodiment, p=1 and q=0 and at least R₃, R₇, and R₁₂independently include a cationic substituent attached to the fused ringsystem and R₁₇ is a hydrophobic substituent that includes a hydrophobicgroup selected to give the ceragenin compound its desiredhydrophobic/hydrophilic characteristics, which allows the ceragenincompound to non-covalently bond to a polymer and elute out over timeand/or be selectively exposed to microbes. The R₁₇ substituent may behydrophobic but still include one or more heteroatoms (O or N) by havingsufficient number of carbon atoms attached thereto to form a hydrophobicgroup. The hydrophobic group may be branched, substituted, orunsubstituted and the branching may occur at the heteroatom (e.g.,dialkyl amines). The hydrophobic substituent is preferably attached atR₁₇ when q=0 and R₁₈ when q=1, but may be attached at other locations onthe D ring or on R groups at locations on rings A, B, or C of Formula I.Where a hydrophobic substituent has a hydrophobic group attached to aheteroatom of an alkyl group, the hydrophobic group may have from 1-20carbons, preferably 8, 9, 10, 11, 12, 13, 14, 15 or more carbons and 20,18, 17, 16, 15 or fewer carbons or within a range thereof. Thehydrophobic group may also include a hydrophobic moiety such astrimethylsilane. The hydrophobic group may include one or more alkylgroups each having 4 or more, 6 or more, 8 or more, 10 or more or 12 ormore carbons. The hydrophobic group can be attached to the sterolstructure by an alkyl group linking to the heteroatom. The linkage maybe an ester, an ether, an amine, or an amide. Ester linkages arepreferred where hydrolysis is desired and/or no charge is desired toimpart greater hydrophobicity. There the heteroatom includes an amine,the hydrophobic group is preferably a dialkyl. Examples of a suitablehydrophobic substituents having a hydrophobic group as described hereinare C₁₃-alkylamino-C₅-alkyl and di-(C₁-C₂₀) alkylamino-(C₁-C₁₀)-alkyl,which can be covalently bonded to the D ring at R₁₇ or R₁₈ (Formula I).

A number of examples of compounds of Formula I that may be used in theembodiments described herein are illustrated in FIG. 1B. Suitableexamples of hydrophobic ceragenins useful in producing a compositionthat will selectively elute from a polymer include, but are not limitedto, CSA-131, CSA-132, CSA-133, CSA-134, CSA-135, CSA-137, CSA-138,CSA-144, and CSA-145. The foregoing compounds have a C Log P valuegreater than 6.5, 7.5, 8.5, and in some cases greater than 10.Nevertheless, CSA-135 was found to be less satisfactory compared toother ceragenins disclosed herein having different C Log P valuesrelative to their ability to elute and kill microbes in a desiredmanner. For example, CSA-136 was unexpectedly found to be advantageousover CSA-135 in its ability to elute and kill microbes. This suggeststhat desirable C Log P values between 6.0 and 9.0 may be desirable insome cases.

FIG. 1A illustrates compounds that have a C Log P value less than 6.5.When contrasted with the compounds of FIG. 1B, the compounds of FIG. 1Aillustrate the types of changes that impart hydrophobicity of a C Log Pvalue greater than 6.0, 6.25, 6.5, 7.5, 8.5, or 10. For example, wherethe heteroatom is part of an ester group, a hydrocarbon chain length of9 or greater extending beyond the heteroatom is sufficient to impart thedesired hydrophobicity. Where an amine group is the heteroatom, ahydrocarbon chain length of 11 carbon atoms or greater extending beyondthe heteroatom is sufficient to impart a C Log P value greater than 6.5(with the proviso that CSA-135 was unexpectedly found to be lesssatisfactory than CSA-136 in its ability to elute and kill microbes).Other moieties such as trimethyl silane can be added to allow for aminegroups to be used with shorter chain lengths or to provide additionalhydrophobicity.

With reference again to Formula I, more specifically, each of fusedrings A, B, C, and D is independently saturated, or is fully orpartially unsaturated, provided that at least two of A, B, C, and D aresaturated, wherein rings A, B, C, and D form a ring system; each of m,n, p, and q is independently 0 or 1; each of R₁ through R₄, R₆, R₇, R₁₁,R₁₂, R₁₅, and R₁₆ is independently selected from the group consisting ofhydrogen, hydroxyl, a substituted or unsubstituted (C₁-C₁₀) alkyl,(C₁-C₁₀) hydroxyalkyl, (C₁-C₁₀) alkyloxy-(C₁-C₁₀) alkyl, (C₁-C₁₀)alkylcarboxy-(C₁-C₁₀) alkyl, (C₁-C₁₀) alkylamino-(C₁-C₁₀)alkyl, (C₁-C₁₀)alkylamino-(C₁-C₁₀) alkylamino, (C₁-C₁₀) alkylamino-(C₁-C₁₀)alkylamino-(C₁-C₁₀) alkylamino, a substituted or unsubstituted (C₁-C₁₀)aminoalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted arylamino-(C₁-C₁₀) alkyl, (C₁-C₁₀) haloalkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C₁-C₁₀) aminoalkyloxy, asubstituted or unsubstituted (C₁-C₁₀) aminoalkyloxy-(C₁-C₁₀) alkyl, asubstituted or unsubstituted (C₁-C₁₀) aminoalkylcarboxy, a substitutedor unsubstituted (C₁-C₁₀) aminoalkylaminocarbonyl, a substituted orunsubstituted (C₁-C₁₀) aminoalkylcarboxamido, H₂N—HC(Q₅)-C(O)—O—,H₂N—HC(Q₅)-C(O)—N(H)—, (C₁-C₁₀) azidoalkyloxy, (C₁-C₁₀) cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, (C₁-C₁₀) guanidinoalkyloxy, (C₁-C₁₀)quaternaryammoniumalkylcarboxy, and (C₁-C₁₀) guanidinoalkyl carboxy,where Q₅ is a side chain of any amino acid (including a side chain ofglycine, i.e., H), P.G. is an amino protecting group, and each of R₅,R₈, R₉, R₁₀, R₁₃, and R₁₄ is independently deleted when one of fusedrings A, B, C, or D is unsaturated so as to WS complete the valency ofthe carbon atom at that site, or selected from the group consisting ofhydrogen, hydroxyl, a substituted or unsubstituted (C₁-C₁₀) alkyl,(C₁-C₁₀) hydroxyalkyl, (C₁-C₁₀) alkyloxy-(C₁-C₁₀) alkyl, a substitutedor unsubstituted (C₁-C₁₀) aminoalkyl, a substituted or unsubstitutedaryl, (C₁-C₁₀) haloalkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, oxo, a linkinggroup attached to a second steroid, a substituted or unsubstituted(C₁-C₁₀) aminoalkyloxy, a substituted or unsubstituted (C₁-C₁₀)aminoalkylcarboxy, a substituted or unsubstituted (C₁-C₁₀)aminoalkylaminocarbonyl, H₂N—HC(Q₅)-C(O)—O—, H₂N—HC(Q₅)-C(O)—N(H)—,(C₁-C₁₀) azidoalkyloxy, (C₁-C₁₀) cyanoalkyloxy, P.G.-HN—HC(Q₅)-C(O)—O—,(C₁-C₁₀) guanidinoalkyloxy, and (C₁-C₁₀) guanidinoalkylcarboxy, where Q₅is a side chain of any amino acid, PG. is an amino protecting group,provided that at least two or three of R₁₋₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆,R₁₇, and R₁₈ are independently selected from the group consisting of asubstituted or unsubstituted (C₁-C₁₀) aminoalkyl, a substituted orunsubstituted (C₁-C₁₀) aminoalkyloxy, (C₁-C₁₀) alkylcarboxy-(C₁-C₁₀)alkyl, (C₁-C₁₀) alkylamino-(C₁-C₁₀) alkylamino, (C₁-C₁₀)alkylamino-(C₁-C₁₀) alkylamino (C₁-C₁₀) alkylamino, a substituted orunsubstituted (C₁-C₁₀) aminoalkylcarboxy, a substituted or unsubstitutedarylamino (C₁-C₁₀) alkyl, a substituted or unsubstituted (C₁-C₁₀)aminoalkyloxy (C₁-C₁₀) aminoalkylaminocarbonyl, a substituted orunsubstituted (C₁-C₁₀) aminoalkylaminocarbonyl, a substituted orunsubstituted (C₁-C₁₀) aminoalkylcarboxyamido, a (C₁-C₁₀)quaternaryammonium alkylcarboxy, H₂N—HC(Q₅)-C(O)—O—,H₂N—HC(Q₅)-C(O)—N(H)—, (C₁-C₁₀) azidoalkyloxy, (C₁-C₁₀) cyanoalkyloxy,P.G.-HN—HC(Q₅)-C(O)—O—, (C₁-C₁₀) guanidinoalkyloxy, and a (C₁-C₁₀)guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof.Additional examples of specific CSA compounds are disclosed inApplicant's copending U.S. application Ser. No. 13/288,902 Filed Nov. 3,2012, which is incorporated herein by reference.

A “ring” as used herein can be heterocyclic or carbocyclic. The term“saturated” used herein refers to the fused ring of Formula I havingeach atom in the fused ring either hydrogenated or substituted such thatthe valency of each atom is filled. The term “unsaturated” used hereinrefers to the fused ring of Formula I where the valency of each atom ofthe fused ring may not be filled with hydrogen or other substituents.For example, adjacent carbon atoms in the fused ring can be doubly boundto each other. Unsaturation can also include deleting at least one ofthe following pairs and completing the valency of the ring carbon atomsat these deleted positions with a double bond; such as R₅ and R₉; R₈ andR₁₀; and R₁₃ and R₁₄.

The term “unsubstituted” used herein refers to a moiety having each atomhydrogenated such that the valency of each atom is filled.

The term “halo” used herein refers to a halogen atom such as fluorine,chlorine, bromine, or iodine.

Examples of amino acid side chains include but are not limited to H(glycine), methyl (alanine), —CH₂—(C═O)—NH₂ (asparagine), —CH₂—SH(cysteine), and —CH(OH)—CH₃ (threonine).

An alkyl group is a branched or unbranched hydrocarbon that may besubstituted or unsubstituted. Examples of branched alkyl groups includeisopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl,tert-pentyl, isohexyl. Substituted alkyl groups may have one, two, threeor more substituents, which may be the same or different, each replacinga hydrogen atom. Substituents are halogen (e.g., F, CI, Br, and I),hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protectedcarboxy, cyan, methylsulfonylamino, alkoxy, acyloxy, nitro, and lowerhaloalkyl.

The term “substituted” used herein refers to moieties having one, two,three or more substituents, which may be the same or different, eachreplacing a hydrogen atom. Examples of substituents include but are notlimited to halogen (e.g., F, CI, Br, and I), hydroxyl, protectedhydroxyl, amino, protected amino, carboxy, protected carboxy, cyan,methylsulfonylamino, alkoxy, alkyl, aryl, aralkyl, acyloxy, nitro, andlower haloalkyl.

An aryl group is a C₆₋₂₀ aromatic ring, wherein the ring is made ofcarbon atoms (e.g., C₆-C₁₄, C₆₋₁₀ aryl groups). Examples of haloalkylinclude fluoromethyl, dichloromethyl, trifluoromethyl,1,1-difluoroethyl, and 2,2-dibromoethyl.

An aralkyl group is a group containing 6-20 carbon atoms that has atleast one aryl ring and at least one alkyl or alkylene chain connectedto that ring. An example of an aralkyl group is a benzyl group.

A linking group is any divalent moiety used to link one compound toanother. For example, a linking group may link a second compound to acompound of Formula I. An example of a linking group is (C₁-C₁₀)alkyloxy-(C₁-C₁₀) alkyl.

Amino-protecting groups are known to those skilled in the art. Ingeneral, the species of protecting group is not critical, provided thatit is stable to the conditions of any subsequent reaction(s) on otherpositions of the compound and can be removed at the appropriate pointwithout adversely affecting the remainder of the molecule. In addition,a protecting group may be substituted for another after substantivesynthetic transformations are complete. Clearly, where a compounddiffers from a compound disclosed herein only in that one or moreprotecting groups of the disclosed compound has been substituted with adifferent protecting group, that compound is within the disclosure.Further examples and conditions are found in T. W. Greene, ProtectiveGroups in Organic Chemistry, (1st ed., 1981, 2nd ed., 1991).

A person of skill will recognize that various ceragenin compoundsdescribed herein preserve certain stereochemical and electroniccharacteristics found in steroids. The term “single face,” as usedherein, refers to substituents on the fused sterol backbone having thesame stereochemical orientation such that they project from one side ofthe molecule. For example, substituents bound at R₃, R₇ and R₁₂ ofFormula I may be all β-substituted or α-substituted. The configurationof the moieties R₃, R₇ and R₁₂ may be important for interaction with thecellular membrane.

Compounds include but are not limited to compounds having cationicsubstituents (e.g., amine or guanidine groups) covalently attached to asterol backbone or scaffold at any carbon position, e.g., cholic acid.In various embodiments, a group is covalently attached at any one ormore of positions R₃, R₇, and R₁₂ of the sterol backbone. In additionalembodiments, a group is absent from anyone, or more, of positions R₃,R₇, and R₁₂ of the sterol backbone.

Other ring systems can also be used, e.g., 5-member fused rings.Compounds with backbones having a combination of 5- and 6-membered ringsare also contemplated. Cationic functional groups (e.g., amine orguanidine groups) can be separated from the backbone by at least one,two, three, four or more atoms.

Ceragenins with hydrophobic substituents can be prepared using thetechniques described in Applicant's U.S. Pat. No. 6,767,904, with themodification being using longer chain alkyls to form a more hydrophobicsubstituent. For example, instead of using an octyl amine to form thefunctional group at R₁₇, a corresponding longer chain amine can be used.

II. Non-Covalent Incorporation of Ceragenins into a Polymer

Hydrophobic ceragenin compounds incorporated into a polymer can benon-covalently associated with the polymer. Upon contact with moisture,the ceragenin can leach or elute from the polymer. Ceragenins aregenerally soluble in water, and ceragenins Scan be associated withpolymers to control release rates. Selection of appropriate polymer andceragenin structures allows for an extended period of release of theceragenin.

For example, the chain extending from a heteroatom (e.g., N) on R₁₇(Formula I) can be tailored to allow varied rates of elution from ahydrogel polymer. Exemplary chains included, lipids, hydrophobic chains(e.g., aliphatic), hydrophilic (e.g., polyethyleneoxide), or any chainthat interacts with the polymer is a way that allows modification of therate of elution. Longer chain lengths will retain the ceragenin withinthe polymer matrix (in particular the hydrophobic domains). In oneembodiment, the ceragenin compound may have a carbon chain of at least 9carbons attached to the D ring of the sterol group (Formula I). Forexample, the carbon chain of at least 9 carbons may be attached to R₁₇group of Formula I, or the C₂₄ carbon or other similar carbon of asterol backbone.

The particular ceragenins incorporated into the polymer may be solubleor partially soluble in aqueous solutions. Additionally, ceragenins whenblended with the water and the appropriate surfactant can be handled inthe form of gels, or emulsions. Block copolymers based on ethylene oxideand/or propylene oxide, in particular, Pluronic-type surfactants, areespecially useful for this purpose. Pluronic is a product of BASF, abusiness with offices in Port Arthur, Tex., USA.

Ceragenin compounds can be incorporated into a polymer at any suitablestep during manufacture of a hydrogel material or product. For example,in an embodiment, a polymer can be brought into contact with a solutionof ceragenins by immersion, spraying, printing, or coating, etc.Suitable solvents include short chain alcohols such as ethanol,methanol, isopropyl alcohol, and the like. If desired, the solvent usedto incorporate the ceragenin can be removed, for example, byevaporation. If necessary the polymer can be dried by utilizing forcedhot air, oven drying, air at room temperature, microwave drying, or theuse of heated drying drums, vacuum chambers, etc. In some manufacturingsystems the normal air flow and temperature sufficiently dry thesubstrate without a discrete drying process.

Ceragenin compounds are known to be soluble in water. Alternatively,ceragenin compounds are also soluble in such materials as ethanol (andother alcohols), propylene glycol, glycerine, and polyols, or mixturesthereof with or without water can be used in incorporate ceragenincompounds into a polymeric material. Furthermore ceragenins can beincorporated as gels, emulsions, suspensions, and in dry form.

In another embodiment ceragenin is incorporated into a polymer duringpolymerization of the monomers. In these processes, the ceragenin can beincluded in the monomer blend during polymerization. The ceragenin infinal polymer can be noncovalently incorporated into the polymer andwill accordingly elute when contacted with a solvent such as water.

III. Elution

When the ceragenin compound is incorporated into a polymeric material,the hydrophobicity/hydrophilicity of the polymer and the ceragenincompound are selected to cause the ceragenin compound to non-covalentlybond to the hydrogel polymer. The non-covalent bonding prevents theceragenin compound from being released all at once in the presence of asolvent. Rather, the bonding allows the ceragenin compound to bereleased over time in the presence of a solvent.

The non-covalent bonding depends on the composition of both the polymerand the ceragenin and therefore need to be selected together to producethe desired elution. The selection is typically carried out by selectinga particular polymer having desired chemical and mechanical propertiesfor a particular application. For example, if the polymer is coated on amedical device to be implanted in vascular tissue, the polymer isselected for compatibility with vascular tissue and blood. If thepolymer is used to form a contact lens, the polymer is selected for itscompatibility with the eye and the need to form the polymer in a shapethat will correct vision. The hydrophobicity/hydrophilicity of thepolymer material is therefore somewhat constrained by the particularapplication.

The ceragenin compound has a hydrophobicity selected to providenon-covalent bonding to the particular polymer. The ceragenin may beselected to have R groups that bond non-covalently the functional groupsof the polymer. For example, a polyacrylate based polymer may have acertain percentage of hydrophobic groups and hydrophilic groups in thepolymer matrix and the ceragenin compound may be selected to have ahydrophobic R₁₇ substituent (where q=0 in Formula I) that non-covalentlybonds to the hydrophobic groups of the polymer to cause a relativelyconsistent elution over a period of days or weeks.

In some cases, the solvent may also influence elution. In oneembodiment, the solvent is water. In some embodiments, the solvent maybe saline.

In one embodiment, the hydrogel polymer and the ceragenin compound areselected to yield non-covalent bonding that provides a release rate of0.1-100 μg/ml, 0.5-50 μg/ml, or 1-10 μg/ml at three days, one week, orone month in water or saline. In one embodiment, the foregoing elutionrate remains within the foregoing ranges for at least 3 days, one week,or one month. These elution rates are achieved in part by thenon-covalent bonding that prevents rapid release of the compound, whichresults in more compound being available at a later date.

As mentioned above, it has been surprisingly found that non-covalentlybound ceragenins in hydrogels selectively elute in the presence ofmicrobes. This is a surprising and unexpected result that makes the useof polymer-ceragenin compounds particularly advantageous as compared toother materials, such as ceragenins covalently bonded to the surface ofa polymer.

Those skilled in the art will recognize that the selection of theparticular polymer and ceragenin compound will depend on the particularapplication and the appropriate selection can be made by one of skill inthe art using the teachings and examples provided herein.

IV. Hydrogel Polymers

One type of polymer that is particularly useful for incorporatinghydrophobic ceragenin compounds are hydrogel polymers.

Examples of suitable hydrogel polymers include, but are not limited to,polyvinyl alcohol, sodium polyacrylate, acrylate polymers, polyethyleneoxide, poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (polyAMP S),polyvinylpyrrolidone, polyacrylamide, silicone, agarose,methylcellulose, hyaluronan, hydrolyzed polyacrylicnitrile, combinationsof these. The hydrogels may be copolymers. The copolymers may includehydrophobic and hydrophilic units.

In one embodiment, the hydrogel is suitable for manufacturing a contactlens. Hydrophilic contact lenses can be formed from cross-linkedpolymers based on hydrophilic derivatives of acrylic or methacrylicacid, hydrophilic vinylic monomers such as vinylpyrrolidone, and thelike. The hydrogels preferably include hydrophobic regions made fromblocks or monomers that are hydrophobic.

An example of a suitable contact lens hydrogel is disclosed in U.S. Pat.No. 8,011,784, which is incorporated herein by reference.

The hydrogel polymers may be formed into a contact lens having a shapeand structure suitable for correcting vision. Those skilled in the artare familiar with the shapes and structures of hydrogel polymers thatcan provide correction for vision. Other devices that can be formed fromthe hydrogels include wound healing devices such as tissue scaffolds andwound dressing.

V. Medical Devices and Coatings

The polymers described herein may be used in various applications,including, but not limited to, medical devices, coatings, bandages,implants, tissue scaffolding, and the like. FIG. 2 is a schematicrepresentation of a medical device 100 that includes a substrate 110 anda polymeric coating 120.

The substrate 110 may be made of any material suitable for supportingand/or adhering to a hydrogel material. The substrate can be polymeric,metallic, an alloy, inorganic, and/or organic. In one embodiment, thesubstrate is a biocompatible or bioabsorbable material. Suitablebiocompatible metallic materials include, but are not limited to,stainless steel, tantalum, titanium alloys (including nitinol), andcobalt alloys (including cobalt-chromium-nickel alloys). Suitablenonmetallic biocompatible materials include, but are not limited to,polyamides, polyolefins (i.e. polypropylene, polyethylene etc.),nonabsorbable polyesters (i.e. polyethylene terephthalate), andbioabsorbable aliphatic polyesters (i.e. homopolymers and copolymers oflactic acid, glycolic acid, lactide, glycolide, para-dioxanone,trimethylene carbonate, ε-caprolactone, and the like, and combinationsof these)

The thickness of the substrate will depend on the device and thematerial but may be 0.1, 1.0, 10 mm or greater and/or 100, 10, or 1 mmor less and/or within a range thereof.

The thickness of polymeric coating 120 is generally less than thethickness of substrate 110. Polymeric coating 120 may have a thicknessof 0.01, 0.1, 1.0, or 10 mm or greater and 100, 10, 1.0, or 0.1 mm orless or within a range thereof.

The polymeric coating 120 can be continuous or non-continuous. Thecoating may be applied to the substrate using techniques such as dipcoating, spin coating, or the like.

Examples of medical devices that can be formed from a polymer containinghydrophobic ceragenin compounds or can have such a polymer coatedthereon include but are not limited to bone implants, bone pins, bonescrews, tissue grafts, airway devices such as endotracheal tubes,implantable devices such as coronary stents, peripheral stents,catheters, arterio-venous grafts, by-pass grafts, pacemaker anddefibrillator leads, anastomotic clips, arterial closure devices, patentforamen ovale closure devices, and drug delivery balloons. The polymermay be coated on or form any portion of the structures of such devicesand is preferably on an outer surface and more preferably on an outservice that contacts tissue or a tissue air interface (when the deviceis implanted).

VI. Stabilization of Ceragenins by pH

In one embodiment a ceragenin compound can have hydrolysable linkagesthat attach the cationic substituents to the sterol group (e.g., esterbonds). Hydrolysis of these linkages inactivates the ceragenin. To makethe ceragenin stable, an acid can be added to achieve a pH less than 6,5.5, 5, or 4.5 and optionally greater than 2, 2.5, or 3 or a rangethereof. Stability before use is important to give a desired shelf lifeand instability during and after use can be desirable to prevent longterm accumulation of ceragenins in biological systems.

It may be advantageous to adjust the degree of neutralization of thepolymer to improve the stability of the ceragenin. The degree ofneutralization of the polymer can be adjusted during its manufacturingprocess, or subsequently. Alternatively, the ceragenin can be suspendedor dissolved in an acidic solution; and when the ceragenin suspension orsolution is added to the hydrogel polymer the degree of neutralizationof the hydrogel would thereby be adjusted.

VII. Examples

To better understand the mechanism by which hydrophobic ceragenincompounds can prevent bacterial colonization, the bonding betweenCSA-138 and a hydrogel used in contact lenses was evaluated. In a firstexample, we determined the rate at which CSA-138 elutes from a hydrogelsuitable for use in contact lenses. To quantify the amount of ceragenineluting from the hydrogel, we used LC/MS using a mass-labeled internalstandard. However, this method only gave detection limits of about 2μg/ml, and we were able to effectively kill bacteria at constant elutionrates below the detection limit. For example, the elution fell belowdetection limits within five days of elution from lenses in whichCSA-138 had been incorporated at 1%, yet the ceragenins appeared tostill be providing suitable kill rates.

To decrease the detection limit for CSA-138, we prepared a radiolabeledversion of CSA-138 (CSA-138T2), incorporated it into contact lenses, andquantified its elution from lenses using scintillation counting.

Example 1

Lenses containing 1% CSA-138 were stored in 0.5 mL phosphate bufferedsaline (PBS) prior to testing. One set of lenses was autoclaved for 45min before elution studies were performed. For elution studies, lenseswere suspended in 2 ml aliquots of PBS, 10% TSB growth medium, 10% TSBgrowth medium containing 10⁶ CFU of Staphylococcus aureus, or 10% TSBgrowth medium containing 10⁶ CFU of Pseudomonas aeruginosa.Corresponding aliquots were exchanged every 24 h, including bacterialinocula. Samples were removed every 24 h and analyzed for the presenceof CSA-138 using scintillation counting. A standard curve was generatedto correlate counts per minute to concentration of CSA-138. Allexperiments were performed in triplicate.

Though some variations from day to day were observed, a recognizabletrend was observed in the elution profile of lenses suspended in PBS(FIG. 3). As expected, the elution on the first day was relatively high(about 2.2 μg/ml). Over the course of following 19 days, daily elutionchanged from approximately 1.6 to 1.4 μg/ml per day.

A comparable elution profile was observed with lenses that wereautoclaved prior to the start of the study, except that the initialamount of material that eluted decreased somewhat (FIG. 4). Thisdecrease in elution is likely due to enhanced elution into the storagesolution during the autoclaving process. Over the course of the study(from day 2 to 20), the amount of CSA-138 that eluted changed fromapproximately 1.4 to 1.2 μg/ml per day.

It was anticipated that an increase in the osmolality of an aqueoussolution would decrease the solubility of CSA-13 and slow elution. Wedetermined the elution profile in 10% TSB in PBS, and as expectedelution decreased (FIG. 5) to match that seen with lenses that had beenautoclaved.

Because kill rates appeared to be happening at such low concentrations,we hypothesized that the presence of bacteria was influencing theelution of CSA-138 from lenses. To test this hypothesis, lenses wereincubated with S. aureus or P. aeruginosa and elution was monitored.These experiments were performed for nine and eight days, respectively.Elution of CSA-138 fluctuated substantially and to a much greater extentthan outside of the presence of bacteria (FIGS. 6 and 7). Because ofthese variations, the experiments were shortened relative to elutionexperiments without bacteria. Though there was substantial variation inthe elution in the presence of bacteria, it was possible to determinethe significance of the differences in elution comparing samples withand without bacteria. After the first day, differences gave a p value of0.05 and for many of the days, the p value was below 0.01. These resultsargue that bacteria impact elution of CSA-138 from lenses.

The MIC values of CSA-138 for S. aureus and for P. aeruginosa are 0.5and 1.0 μg/ml, respectively. The elution of CSA-138 from lenses givesconcentrations that are just able to eliminate the inocula introduced.Autoclaving the lenses, increasing the osmolality in the surroundingsolution, and the presence of bacteria impact the elution profilemodestly.

If one takes the elution profile given in FIG. 5 and extends the trenduntil elution of CSA-138 drops below 1 μg/ml, this would require about40 days (elution decreases from 1.4 to 1.2 μg/ml per day between daystwo and 20; a decrease from 1.2 to 1.0 μg/ml per day would be expectedto require another 19 days). Thus, it would be expected that elution ofCSA-138 would be sufficient to eliminate reasonable inocula of bacteriafor as many as 40 days. As noted in a previous report, elution ofCSA-138 from lenses prevents colonization by S. aureus for 30consecutive days and by P. aeruginosa for 19 days. These studies areperformed with relatively high inocula (10⁶ CFU), and it is anticipatedthat CSA-138 eluting after 30 days would be sufficient to eliminatesmaller inocula.

Optimization of the structure of CSA-138 has yielded a potentantimicrobial agent that associates with contact lens material andelutes at the concentration necessary to eliminate substantial inoculaof Gram-positive and -negative bacteria. Considering the number ofbacteria to which lenses are typically exposed, it is likely that lowerconcentrations of CSA-138 could be used while continuing to preventbacterial growth on lenses.

For purposes of this invention, “physiological conditions” are aqueousconditions where the pH, temperature, and salt concentrations aregenerally suitable for sustaining life (e.g., for many, but not alldevices, physiological conditions is often a pH near 7, temperaturesnear 37° C., and salt concentration near 150 mM).

The present invention may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the invention is, therefore, indicatedby the appended claims rather than by the foregoing description. Allchanges which come within the meaning and range of equivalency of theclaims are to be embraced within their scope.

What is claimed is:
 1. A cationic steroidal anti-microbial (CSA)compound selected from the group consisting of:

and salts thereof.
 2. The CSA compound of claim 1, wherein the CSAcompound is a hydrochloride salt.
 3. A cationic steroidal anti-microbial(CSA) compound selected from the group consisting of:

and salts thereof.
 4. A cationic steroidal anti-microbial (CSA) compoundselected from the group consisting of:

and salts thereof.
 5. A device comprising a polymer structure and theCSA compound of claim 1 incorporated into the polymer structure.
 6. Thedevice of claim 5, wherein the CSA compound is incorporated into thepolymer structure with non-covalent interactions.
 7. The device of claim5, wherein the polymer structure comprises polyvinyl alcohol, sodiumpolyacrylate, an acrylate polymer, polyethylene oxide, polyAMPS,polyvinylpyrrolidone, polyacrylamide, silicone, agarose,methylcellulose, hyaluronan, or a combination thereof.
 8. The device ofclaim 5, wherein the device comprises a medical device.
 9. The device ofclaim 8, wherein the medical device is selected from the groupconsisting of bone implant, bone pin, bone screw, tissue graft,endotracheal tube, coronary stent, peripheral stent, catheter,arterio-venous graft, by-pass graft, pacemaker or defibrillator lead,anastomotic clip, arterial closure device, patent foramen ovale closuredevice, and drug delivery balloon.
 10. The device of claim 5, whereinthe polymer structure includes a polymer coated on a substrate.
 11. Thedevice of claim 5, wherein the hydrophobic CSA compound elutes from thepolymer structure in excess saline water at a rate of 0.1-100 μg/ml at 3days and/or over a period of 3 days.
 12. The device of claim 5, whereinthe hydrophobic CSA compound elutes from the polymer structure in excesssaline water at a rate of 0.5-50 μg/ml at one week and/or over a periodof one week.
 13. The device of claim 5, wherein the hydrophobic CSAcompound elutes from the polymer structure in excess saline water at arate of 1-10 μg/ml at one month and/or over a period of one month.